Study On The Relatioinship Between The Increased Transduction Activity Of Wnt/β-catenin Signal And COX-2 In BMP9-induced Osteogenic Differentiation Of MEFs

Bone non-union and bone defect are the most common bone-related diseases.However,the current strategies for these disease are not so effectively.To date,engineering bone is the best way for the treatment of the above mentioned bone diseases.However,the tissue engineering bone still need to explore the ideal scaffold,seed cells and osteogenic inducing factors.BMP9 is a promising factor of bone tissure engineering because it possesses efficicaous potential to induce osteogenesis in multiple progenitor cells(MPCs).In this study,we scheduled to further dissect the mechanism underlying the BMP9-induced osteogenesis.RT-PCR,Western blot,cytoimmunofluorescence staining,histochemical staining,Alizarin Red S staining,cell cycle analysis,luciferase reporter assay and ChIP assay were introduced for the related detection.Firstly,we confirmed the osteogenic inducing capability of BMP9 in MEFs.And then,we analyzed the effect of BMP9 on the expression of COX-2,as well as the relationship between the effect of COX-2 on BMP9-induced osteogenic differentiation and PGE2/EPs pathway.Finally,we analyzed the possible relationship between the effect of COX-2 on BMP9-induced osteogenic differentiation and the activiation of Wnt/β-catenin signal in stem cells.The results showed that BMP9 can induce osteogenic differentiation apparently,and increase the expression of COX-2.Inhibition of COX-2 attenuates the BMP9-induced osteogenic differentiation.BMP9 increases the level of PGE2 in MEFs.Inhibitor of EP2 or EP4 both can decrease the BMP9-induced osteogenic differentiation in MEFs,and the effect of EP4 inhibitor is more pronounced.The effect of COX-2 on the BMP9-induced osteogenic differentiation may not be related with the inhibition of proliferation in the stem cells.BMP9 increases the transduction activity of Wnt/β-catenin signal,which can be enhance by exogenous COX-2,but decreased by COX-2 specific inhibitor.Inhibitor of EPs or cAMP can also decrease the effect of BMP9 on the transduction of Wnt/β-catenin signal.The effect of COX-2 on Wnt/β-catenin signal may be mediated by increasing the phosphorylation of CREB,which can regulate the expression of β-catenin directly.The inhibtory effect of COX-2 specific inhibitor on the osteogenic differentiation of stem cells may be diminished or reversedbyATRA.Ourfindings suggested that COX-2 and Wnt/β-catenin signal may form a feedback regulatory loop to orchestrate the BMP9-induced osteogenic differentiation in MPCs.